Various common diseases of the cardiovascular system like venous and arterial thrombosis, pulmonary embolism, myocardial infarction and related unstable angina are currently treated with parenteral administration of unfractionated heparin (UFH) or its lower molecular weight analogous (LMWH) in clinics.[1] Despite the development of more conveniently administered oral anticoagulants subcutaneously injected heparin is still the benchmark anticoagulant in prophylaxis of patients with risk of venous thromboembolism after surgery. However, after several decades of successful clinical use of UFH and LMWH as parenteral anticoagulants during surgical procedures as well as prophylaxis of thrombotic complications, bleeding still remains one of the major complications.[2] In addition, the fact that both UFH and LMWHs are derived from animal tissues raises concerns over their safety as it could lead to severe risk of disease transmission.[3]
Heparin is a naturally occurring, partially O- and N-sulfated linear polysaccharide with broad structural variability. It belongs to the class of glycosaminoglycans consisting of an alternating sequence of D-glucosamine and uronic acids. It is commonly isolated from mucosa of porcine intestine, but is also naturally found in lung and liver tissue. Its natural dispersity is broad and molecular weights of the isolated material range from around 4 to 30 kDa with an abundance maximum at 15 kDa. Lower molecular weight heparins (LMWHs) refer to the same composition and structural variability but are of lower molecular weight. Such LMWHs are refined from UFH by chemical or enzymatic degradation which yields, e.g. Enoxaparin (4.5 kDa) or Tinzaparin (6.5 kDa) with lower polydispersity compared to UFH. FIG. 1 illustrates the polydispersity and molecular weight range in daltons of commonly applied UFH and LMWH. The graph is taken from reference [1]. The respective molecular weight and dispersity characteristic of heparin determines its mode of action as an anticoagulant as well as its bioavailability and pharmacokinetics (vide infra).
Recently, adverse immunogenic reactions due to antibody development against heparins have been observed in patients after heparin administration that can lead to life threatening heparin induced thrombocytopenia (HIT).[4] HIT is characterized by a sudden drop of platelet counts in patients, usually between day 5 and day 13 after the first heparin administration and leads to an increased propensity to thrombosis. It is known, that the electrostatic interaction and binding of heparin to platelet factor 4 (PF4) in plasma induces a conformational change of PF4 which now exposes an epitope that can be recognized by platelet-activating antibodies of the immunoglobulin G class. A minimum of 12-14 saccharide units are required to form such antigenic multi-molecular heparin/PF4 complexes. Therefore, patients with LMWH treatment are less prone to but not safe from HIT development. The ionic complex formation between anionic heparins and positively charged PF4, however, is independent of the composition of the polysulfate since it was shown that non-carbohydrate based anionic, linear polymers can also form complexes with PF4, given the chain length is long enough, thus the molecular weight is high enough, to span the tetrameric PF4 protein.[5] Another unwanted side effect of heparin is osteopenia which is caused by heparin binding to osteoblast cells and results in the release of osteoclast activating factors which in turn causes lowering of bone mineral density.[6]
Especially in such cases of acute, unexpected side effects or also when unintendedly overdosed an antidote for the anticoagulant drug is required. Protamine sulfate, a highly cationic peptide that binds and neutralizes the anionic charges of UFH and LMWHs, and completely reverses the action of UFH and partially reverses the anticoagulant activity of LMWHs. [7]
Due to the huge structural inhomogeneities of heparin including broad molecular weight distribution, the biological and anticoagulant activity of each batch of heparin is unpredictable and different. For standardization the activity of UFH or LMWH is usually given in international units with respect to their activated Factor X (FXa) inhibition. In general, only 30% of the mass fraction of heparin has a strong anticoagulant effect the remaining 60% are biologically inactive in terms of anticoagulation. In addition, the response of each patient towards intravenous (i.v.) heparin administration can be very different, does not show a linear dose response and thus requires constant monitoring in hospital and readjustment of dose during heparin therapy in order to reduce the risk of bleeding. Typically, the activated partial thromboplastin time (aPTT), an in vitro blood coagulation assay with fresh, citrated platelet poor plasma (PPP) of the patient is used in hospitals to monitor and evaluate efficacy of the therapy.
The low molecular weight fraction of heparin binds to circulating antithrombin III (ATIII) with high affinity in the blood stream and thereby causes a conformational, irreversible change in ATIII. ATIII is a natural inhibitor of the two major coagulation enzymes, Factor Xa (FXa) and thrombin (Factor IIa (FIIa)) that circulate in blood. Binding of heparin to ATIII potentiates the affinity of antithrombin for thrombin and FXa due to the induced conformational change in ATIII and yields an up to 1000 times faster binding/inhibition of the enzymes via ternary complex formation. The corresponding reaction schemes are depicted in FIGS. 2A to 2C that show the mode of action of UFH (A), LMWH (B) and the ATIII specific pentasaccharide unit of UFH (C) in ATIII mediated thrombin (FIIa) and FXa inhibition. These Figures are taken from reference [8].
As illustrated in FIG. 2A a minimum chain length of 18 saccharide units of the anticoagulant (as present in UFH) is required in order to efficiently inhibit thrombin since the ATIII specific pentasaccharide unit binds to ATIII with high affinity and induces the conformational change while the remaining 13 units bind to an exosite on thrombin to bridge and stabilize the formed ternary complex (FIG. 2A).[2] Via a similar ternary complex FXa is inhibited by UFH, however, no additional binding of UFH to FXa is required for efficient inhibition. Thus, the preferred inhibition pathway of shorter chain LMWHs within the coagulation cascade is ATIII mediated FXa binding rather than indirect FIIa binding (FIG. 2B). Often the chain length of LMWH is too short in order to form the ternary LMWH/ATIII/FIIa complex. Consequently, the ATIII specific pentasaccharide unit of UFH, is exclusively able to inhibit FXa as illustrated in FIG. 2 C). FIGS. 2A to 2C correspond to according Figures of reference [8].
The pharmacokinetics of UFH are complex mainly due to the structural inhomogeneity of the sulfated polysaccharide and show significant difference from LMWH. Also the specific activity in terms of anticoagulation varies significantly for both and yields unpredictable and non-linear dose response in patients. When administered intravenously UFH and its derivatives immediately bind to several plasma proteins (not only ATIII), the endothelium, platelets or macrophages due to their high negative charge density, which drastically reduces their bioavailability. Thereby UFH shows higher affinity for plasma proteins and cellular blood components than LMWH. After cellular binding heparin becomes internalized into the cells where it is depolymerized. This event is commonly attributed to the rapid saturation phase of clearance. Once the cellular binding sites are saturated, heparin circulates systemically and is cleared more slowly via the kidneys. The non-linear response of UFH after i.v. administration at therapeutic dose is obvious via the observed half lives of 30, 60, and 150 minutes with a bolus of 25, 100, and 400 U/kg, respectively. [9] The pharmacokinetics of LMWHs is superior to the one of UFH since the shorter chains bind less efficiently to plasma proteins and to the endothelium and hence have longer half-lives in plasma and a more linear dose response compared to UFH. Clearance mainly occurs renal and is only an issue for patients with renal disorders. However, protamine sulfate as the only approved and commonly used antidote for UFH is not 100% effective for LMWHs which thus leads to a non-complete reversal of the anticoagulant properties of LMWHs with protamine sulfate as compared to UFH.
A safer and clinically more predictable alternative to UFH and LMWH is fondaparinux, a fully synthetic pentasaccharide drug with high negative charge density. Fondaparinux is an analog of the minimum structural pentasaccharide fraction of heparin for efficient binding to ATIII which exhibits the most linear and predictable dose response among the three mentioned sulfated polysaccharide anticoagulants with a half-life of 17 h after subcutaneous administration. However, fondaparinux is only available via tedious multistep synthesis which makes it cost intensive. In addition, the drug suffers from the lack of an effective clinical antidote as compared to UFH and LMWHs.
Hence, to overcome limitations of current indirect, ATIII mediated anticoagulants the demand for new, more defined, and safer alternatives is emerging. In pursuit of such alternatives, direct thrombin inhibitors such as FDA approved hirudin and argratroban have been developed which, however, are still associated with certain risk of bleeding.[10] Aptamers, small nucleic acid molecules, in contrast, also work as direct inhibitors but are not yet approved. They seem to have no risk of associated bleeding, low immunogenicity, show predictable dose response, adjustable pharmacokinetics, and have an effective antidote available.[11] High production costs remain a disadvantageous fact.
Many other polysulfated or polyanionic polymers have been identified as polymers with anticoagulant properties via in vitro coagulation assays (e.g. aPTT) with platelet poor plasma (PPP).[12] Only a few of them, however, have been proven to work in whole blood as well, e.g. via thromboelastography in vitro, which can measure the time dependent built up and break down of a blood clot in whole blood simultaneously with clot strength. Especially biocompatibility of such polymers including complement activation and cell toxicity is an issue for safe use in vivo.
Besides the therapeutic or prophylactic use of anticoagulants in clinics, there is also a demand for the modification of medical devices which come in contact with blood such as blood bags, blood collection vials, blood based diagnostic assay surfaces and others. Material surface induced thrombus generation is a major clinical concern associated with medical devices such as coronary stents, heart valves, catheters, vascular grafts, extracorporeal tubing, hemodialysis membranes and glucose sensors. The contact activation pathway of the blood coagulation cascade is thought to be involved in the initiation of such events. In order to prevent material surface induced activation of the coagulation pathway and hence thrombus formation catheters are currently routinely pretreated (washed) with heparin solution. By this noncovalent procedure the intended anti-coagulant layer on the surface is not stable and flushed away easily and therefore heparin can unintendedly enter into the bloodstream and affect blood coagulation.
As mentioned above the key enzyme involved in the activation of blood coagulation is thrombin. Hence, approaches to minimize thrombin generation on the surface is an effective way to reduce surface initiated thrombus formation. The covalent surface attachment of heparin has been proven useful and is currently used in many medical devices. However, immobilized heparin's activity is believed to be dependent on the method of covalent surface attachment. With this respect significant issues still exist. Heparin activity is highly diminished by the current immobilization approaches and end-functionalization of heparin without loss of activity is not trivial. Thus new surface modification approaches to generate anti-thrombotic surfaces will revolutionize in particular vascular implant/device industry.
In 2004 Haag et al. synthesized dendritic polyglycerol sulfate for the first time and studied its effect on in vitro blood coagulation and complement activation.[13] Non-sulfated dendritic polyglycerol as the precursor for the latter compound is a highly bio- and haemocompatible, water soluble polymer.[14-17] The same applies to linear polyglycerol.[17-19] Upon sulfation of the multiple hydroxyl groups which are located on the periphery of the dendritic polymer the resulting polyglycerol sulfate (dPGS) exhibits 30% of the anticoagulant activity of UFH in PPP via aPTT in vitro.[13] In addition, dPGS was found to be non-activating for complement in this study via a blood based in vitro assay. In 2008, dPGS was shown by Haag and coworkers to be highly effective in inflammatory settings via L- and P-selectin inhibition in vivo and well tolerated by mice up to an i.v. bolus of 10 to 30 mg/kg.[20-21]
Zhongyu Li and Ying Chang: “Synthesis of Linear Polyether Polyol Derivatives As New Materials for Bioconjugation”, Bioconjugate Chem. 20 (2009), pages 780-789 describes linear polyether polyol derivatives that can carry different substituents, amongst them carboxyl groups or tosylate groups. This publication describes in addition different methods of manufacturing such compounds. One such method is a Williamson reaction in which a linear polyether polyol compound is converted with 2-chloroacetic acid so as to obtain carboxymethyl polyether polyol.
Jens Kölller et al.: “Post-polymerization functionalization of linear polyglycidol with diethyl vinylphosphonate”, Chem. Commun. 47 (2011), pages 8148-8150 describes a partial phosphonatization of a linear polyglycidol with diethyl vinylphosphonate in a Michael-type reaction. The resulting compound is—after saponification—a linear polyglycidol carrying a phosphonate group.
WO 2008/015015 A2 describes dendritic polyglycerol sulfonates.